A human pancreatic lipase is significant as a diagnostic marker for a pancreatic disease such as acute pancreatitis, and a reagent for measuring the enzyme activity in serum is commercially available as a laboratory test reagent. The optimum hydrogen ion exponent (pH) at the time of the measurement of the enzyme activity depends on a substrate or a reaction condition to be employed, but is reported to be in a range of about 7.5 to 10 (see, for example, Non Patent Literature 1). Enzymatic characteristics of the human pancreatic lipase include that the reaction occurs on an interface of an emulsion or a micelle containing a substrate, and that a bile acid and a protein having a molecular weight of approximately 1 lkDa designated as a colipase are necessary for activating the enzyme. If a bile acid and a colipase are absent, the active center of the pancreatic lipase is covered with a domain designated as lid meaning a cover and hence cannot come into contact with a substrate. The bile acid influences formation of an emulsion or a micelle. In addition, the conformation of the human pancreatic lipase is changed if the colipase is additionally present, and the active center of the human pancreatic lipase can bound to a substrate, which can activate the human pancreatic lipase. Thus, it is known that a human pancreatic lipase acts on an interface of a complex containing a hydrophobic substrate, a bile salt and a colipase.
In the measurement of the activity of a human pancreatic lipase in a sample, a triglyceride is conventionally used as the substrate. The triglyceride is used in the form of what is called an emulsion in which it is emulsified and dispersed in a buffer solution by force stirring together with gum arabic, polyvinyl alcohol or the like. In measuring the lipase activity by using a triglyceride substrate, for example, a fatty acid produced through a lipase reaction is quantitatively determined by alkalimetry. Alternatively, an attempt has been made to measure a fatty acid released from a triglyceride substrate through a lipase reaction by an enzymatic method, but it is difficult to spectroscopically measure the lipase activity by using an enzyme conjugated system because the triglyceride substrate shows strong turbidity due to the emulsion. Besides, an emulsion substrate has a disadvantage that phase separation easily occurs during storage, and it is difficult to measure the lipase activity with high reproducibility. In order to solve these problems of the methods using a triglyceride substrate, a method in which a diglyceride is used as a substrate (see, for example, Patent Literatures 1 and 2) and a method in which a synthetic substrate is used (see, for example, Non Patent Literature 2) have been developed, and these methods are currently employed in a routine laboratory testing.
As an advantage of using a diglyceride as a substrate, since a diglyceride is more hydrophilic than a triglyceride, the substrate can be made clear when used in combination with a surface active agent, and hence such a substrate can be conveniently used in a general purpose automatic analyzer.
As a diglyceride substrate, 1,2-diglyceride is principally used. It is known that 1,2-diglyceride is changed into 1,3-diglyceride in a molecule through a transfer reaction of an ester bond, and hence it is used with this change suppressed as much as possible. Besides, it has been proposed that a part of 1,2-diglyceride is converted into 1,3-diglyceride, and the resultant mixture is stabilized in the form of a liquid to be used as a substrate for measuring a human pancreatic lipase (see, for example, Patent Literature 3). Here, it has been reported that a measurement method using a diglyceride substrate is influenced by a non-pancreatic lipase such as a lipoprotein lipase or a hepatic lipase. It has been also reported that the measurement method using a diglyceride substrate has a harmful effect that the specificity to a pancreatic lipase is easily lowered because this method is influenced by a non-pancreatic lipase, such as a lipoprotein lipase, or an esterase contained in blood (see, for example, Patent Literature 4). A non-pancreatic lipase such as a human lipoprotein lipase or hepatic lipase is released into blood by heparin. Therefore, the pancreatic lipase activity in blood cannot be accurately measured in a sample to which heparin has been administered. On the other hand, it is regarded that a routine test reagent for measuring human pancreatic lipase activity using a synthetic substrate is not influenced by a non-pancreatic lipase such as a lipoprotein lipase or a hepatic lipase (see, for example, Non Patent Literature 2), but there is a problem in stability of the synthetic substrate in a weak alkaline region corresponding to the optimum pH of the lipase.
As a method for avoiding the influence of a non-pancreatic lipase, it has been reported that the influence of a non-pancreatic lipase is reduced, in using a commercially available kit for measuring lipase activity using 1,2-dilinoleoyl glycerol, that is, a diglyceride, as the substrate, by replacing sodium deoxycholate used as a bile salt with sodium glycocholate (see, for example, Non Patent Literature 3), but the details are unknown. Besides, not only the measurement method for lipase activity using a diglyceride as a substrate but also a measurement method for lipase activity using a triglyceride as a substrate is influenced by a non-pancreatic lipase, and it has been reported also with respect to this case that the influence of a non-pancreatic lipase can be avoided by replacing deoxycholic acid with glycocholic acid (see, for example, Non Patent Literature 4). However, it is still difficult to measure lipase activity with high reproducibility because a triglyceride substrate has disadvantages that it cannot be spectroscopically measured and phase separation is easily caused during storage as described above.